Production of ethanol by microorganisms provides an alternative energy source to fossil fuels and is therefore an important area of current research. The bacteria Zymomonas naturally produces ethanol, and has been genetically engineered for improved ethanol production. Improvements include elimination of competing pathways, utilization of xylose, and better performance in medium containing biomass hydrolysate (for example: U.S. Pat. No. 7,741,119, U.S. Pat. No. 5,514,583, U.S. Pat. No. 5,712,133, WO 95/28476, Feldmann et al. (1992) Appl. Microbiol. Biotechnol. 38: 354-361, Zhang et al. (1995) Science 267:240-243, and US 2009-0203099 A1). The hydrolysate produced from lignocellulosic and cellulosic biomass can provide an abundantly available, low cost source of carbon substrates for biocatalyst fermentation to produce desired products. Biomass hydrolysate typically includes xylose, as well as inhibitors of fermentation.
For economical fermentative production, it is desired that a biocatalyst does not require addition of any costly nutrients to growth and production media. In particular, it is desired that no vitamin supplements be required for seed or production biocatalyst cultures. Zymomonas requires supplementation of pantothenic acid (PA; also pantothenate, vitamin B5, 3-[(2,4-dihydroxy-3,3-dimethylbutanoyl)amino]propanoic acid) in growth medium, being unable to synthesize this nutrient (Seo et al. (2005) Nat. Biotechnol. 23:63-68; Nipkow et al. (1984) Appl. Microbiol. Biotechnol. 19:237-240 and references therein). PA is an important cellular component as it is required for the synthesis of coenzyme-A (CoA), a compound with many important cellular functions. For many animals it is an essential nutrient, while many plants express enzymes for the synthesis of PA.
E. coli is able to synthesize PA and the biosynthetic pathway is known. E. coli genes encoding enzymes of the pathway have been identified. Increased production of pantothenate has been achieved by overexpressing genes in the biosynthetic pathway of microorganisms that naturally produce pantothenate. Disclosed in WO 2003006664 is increasing expression of coding regions in a Bacillus that naturally produces D-pantothenic acid, such as ybbT, ywkA, yjmC, ytsJ, mdh, cysK, iolJ, pdhD, yuiE, dhas, adk, yusH, yqhJ, yqhK, and/or yqh-I for increased pantothenic acid production. In addition, panE, ylbQ, panB, panD, panC, ilvB, ilvN, alsS, ilvC, ilvD, serA, serC, ywpJ, and/or glyA may be increased in expression. U.S. Pat. No. 6,171,845 discloses amplification of nucleotide sequences encoding keptopantoate reductase, in particular panE, in pantothenic acid producing microorganisms. It was shown that the Saccharomyces cerevisiae YRH063c ORF encodes a protein having ketopantoate reductase activity by complementation of a panE-ilvC mutant in E. coli. US 20050089973 discloses producing panto-compounds in microorganisms where existing biosynthetic pathways are manipulated, such as by overexpressing ketopantoate reductase and aspartate alpha-decarboxylase.
US 2005221466 discloses the use of cells with alanine 2,3-aminomutase activity, which converts alpha-alanine to beta-alanine, for production of pantothenate.
There remains a need for creating Zymomonas strains that are able to grow and produce ethanol in the absence of externally supplied PA. These Zymomonas strains may be used to improve and reduce the cost of ethanol production using this biocatalyst.